Activation of chloride (Cl-) currents in the heart can cause important changes in action potential characteristics, which may increase the dispersion of electrophysiological properties and provide the substrate for the occurrence of arrhythmias. In mammalian myocardial cells the P.I and his colleagues have recently discovered a novel volume-regulated Cl- inward rectifier channel (Cl.ir) (Circulation Research 86:e63-e71, 2000). Distinct from all cardiac Cl- channels described previously, Cl.ir is the only channel that exhibits inwardly rectifying current-voltage relationship. The molecular property and the potential physiological role of Cl.ir in the heart, however, are presently not known. The working hypotheses for this proposal are that 1) Cl.ir may be encoded by ClC-2 gene, a member of the large C1C Cl- channel family, since Cl. ir has many biophysical and pharmacological properties in common to those of CIC-2 channel when expressed in heterologous expression systems, and 2) ClC-2 channels are important in the regulation of the electrical activity and cell volume adaptation in the heart. Three specific aims are proposed to address these hypotheses. Aim 1 will determine the molecular localization of ClC-2 and its correlation with functional Cl.ir distribution pattern in different regions in the heart and the cellular and subcellular (sarcolemmal or subsarcolemmal membrane) localization of ClC-2 channel protein. This information will also greatly facilitate our understanding of the physiological role of ClC-2 channels in heart since regulation of cardiac electrical activity is determined by the heterogeneity of ion channel distribution in different regions. Aim 2 will use combined molecular biological, electrophysiological, and pharmacological approaches to identify the molecular identity of cardiac Cl.ir channel. The results from the proposed experiments will determine whether ClC-2 is responsible for native Cl.ir channels and the resulting molecular clones will provide essential tools for the study of the basic features and the regulatory mechanism of native and recombinant ClC-2 channels at molecular, cellular, and single-channel levels. Aim 3 will characterize the contribution of CIC-2 Cl- channels to the modulation of cardiac electrical activity (including resting membrane potential, diastolic membrane potential, action potential duration and repolarization, automaticity, etc.) and the regulation of cell volume in the heart. Since activation of volume- and acidosis-regulated ClC-2 Cl- channels can produce significant effects on action potential duration and automaticity of both conducting and contractile cells, as shown in our new preliminary experimental data, these channels have important clinical significance for several heart diseases, such as arrhythmias, myocardial ischemia, hypertrophy, and congestive heart failure and represent new important targets for the development of new agents against these diseases. This project has significant potential of elucidating the normal physiological and possible pathophysiological role of the newly discovered cardiac ClC-2 channels in the heart.